1. Field of the Invention
The present invention relates to a human CDR-grafted antibody which specifically reacts with the extracellular region of human CC chemokine receptor 4 (hereinafter referred to as “CCR4”) but does not react with a blood platelet and the antibody fragment thereof Furthermore, the present invention relates to a human CDR-grafted antibody which specifically reacts with the extracellular region of human CCR4 but does not have an inhibiting activity of a CCR4 ligand such as TARC or MDC binding to CCR4 and the antibody fragment thereof. Moreover, the present invention relates to a human CDR-grafted antibody which specifically reacts with the extracellular region of CCR4, has a cytotoxic activity and an inhibiting activity of cytokine production by Th2 cells, and comprises a specific complementarity determining region (hereinafter referred to as “CDR”), and the antibody fragment thereof Also, the present invention relates to a DNA encoding the antibody or the antibody fragment thereof Furthermore, the present invention relates to a vector comprising the DNA, and a transformant transformed with the vector. Moreover, the present invention relates to a method for producing the antibody or the antibody fragment thereof using the transformant, and a medicament such as a therapeutic agent, a diagnostic agent and the like, for Th2-mediated immune diseases such as allergic diseases and the like, which comprises using the antibody or the antibody fragment thereof Additionally, the present invention relates to a medicament such as a therapeutic agent, a diagnostic agent and the like, for cancers such as blood cancers, e.g., leukemia and lymphomatosis, which comprises using the antibody or the antibody fragment thereof
2. Brief Description of the Background Art
Various factors such as eosinophils, mast cells, IgE and the like, play a role in allergic diseases such as bronchial asthma. Eosinophils infiltrate into an inflammatory site and release cytotoxic basic proteins such as MBP (major basic protein) or the like by degranulation and the surrounding tissues are damaged by such cytotoxic basic proteins. Mast cells are bound to an antigen immune complex with IgE which is produced by B cells and release histamine so that an immediate allergic reaction is induced. The allergic reaction is controlled by biologically functional molecules such as cytokines, chemokines, and the like, which take part in signal transduction between cells. IL-5 induces differentiation, survival and degranulation of eosinophils. IL-4 induces B cell activation and production of IgE. IgE produced forms an immune complex with the antigen and causes degranulation of mast cells. It has been found that IL-4, IL-13 and the like are also produced by mast cells and contribute to the production of IgE by B cells (Am. J. Respir. Crit. Care Med., 152, 2059 (1995), Immunol. Today, 15, 19 (1994)). Thus, an elaborate cytokine-chemokine network is present among inflammatory cells and keeps complicated balances.
The cytokines and chemokines are produced by helper T cells which express CD4 on the cell surface (hereinafter referred to as “CD4+Th cells”). Actually, it has been found that infiltration of helper T cells is found in the airway inflammation site of bronchial asthma patients, wherein a considerably large number of the T cells are activated and that the severity and airway hypersensitivity of asthma patient correlates with the number of activated T cells, as well as the activated T cells are also increased in the peripheral blood (Am. Rev. Respir. Dis., 145, S22 (1992)).
The helper T cells are classified into Th1 cells and Th2 cells, depending on the kind of cytokine to be produced thereby (Annu. Rev. Immunol., 7, 145 (1989)). Th2 cells produce IL-4, IL-5, IL-13 and the like. The cytokines produced by Th2 cells are Th2 cytokines.
It has been found that an antigen-specific T cell clone isolated from an atopic disease patient releases Th2 cytokines when stimulated in vitro (Proc. Natl. Acad Sci., U.S.A., 88, 4538 (1991)), and Th2 cells are present in bronchoalveolar lavage fluid (hereinafter referred to as “BAL”) and airway mucosa of asthma patients (N. Engl. J Med., 326, 298 (1992), Eur. J. Immunol., 23, 1445 (1993)). Expression levels of IL-4 and IL-5 mRNAs of Th2 cytokines are increased when mRNA expressions of various cytokines in cells in BAL are examined using an allergic inflammation animal model (Clin. Immunol. Immunopathol, 75, 75 (1995)). Also, when Th2 cells are intravenously or intranasaly administered to mice, asthmatic inflammation is induced in the lungs in antigen specific manner (J. Exp. Med., 186, 1737 (1997), J. Immunol., 160, 1378 (1998)) and eosinophilia is observed (J. Immunol., 16 3128 (1998)). Expression of IL-5 is observed in the airway mucous of asthma patients and the skin lesions of atopic dermatitis patients (J. Clin. Invest., 87 1541 (1991), J. Erp. Med., 173, 775 (1991)), and the expression level of IL-5 in the mucous of chronic allergic rhinitis correlates with the expression level of IL-13, and the amounts of serum total IgE and antigen-specific IgE (Therapeutics, 32, 19 (1998)).
Chemokine is a general term for basic heparin-binding proteins which induce chemotoxis and activation of leukocyte, and classified into subfamilies of CXC, CC, C and CX3C depending on the position of the cysteine residues in the primary structure. 16 of chemokine receptors have been identified so far (Curr. Opi. Immunol., 11, 626 (1999)), and it has been shown that expression of each chemokine receptor is different among the leukocytes such as Th 1 cell, Th2 cell or the like (Cell Engineering, 7,1022 (1998)).
Human CCR4 is a G protein coupled seven transmembrane receptor cloned as K5-5 from a human immature basophilic cell line KU-812. The transmembrane regions of CCR4 are considered to be positions 40 to 67, positions 78 to 97, positions 113 to 133, positions 151 to 175, positions 207 to 226, positions 243 to 270 and positions 285 to 308 in the amino acid sequence, the extracellular regions are considered to be positions 1 to 39, positions 98 to 112, positions 176 to 206 and positions 271 to 284 in the amino acid sequence, and the intracellular regions are positions 68 to 77, positions 134 to 150, positions 227 to 242 and positions 309 to 360 in the amino acid sequence (J. Biol. Chem., 270, 19495 (1995)) At first, it was reported that the ligand of CCR4 is MIP-1α (macrophage inflammatory protein-1α), RANTES (regulated on activation normal T-cell expressed and secreted) or MCP-1 (monocyte chemotactic protein) (Biochem. Biophys. Res. Commun., 218, 337 (1996), WO 96/23068). However, it has been found that TARC (thymus and activation-regulated chernokine) produced from stimulated human peripheral blood mononuclear cells (hereinafter referred to as “PBMC”) and thymus cells (J. Biol. Chem, 271, 21514 (1996)) specifically binds to CCR4 (J. Biol. Chem., 272, 15036 (1997)). It has been also reported that MDC (macrophage-derived chemokine) isolated from macrophage (J. Exp. Med., 185, 1595 (1997)), also known as STCP-1 (stimulated T cell chemotactic protein-1) (J. Biol. Chem., 272, 25229 (1997)), binds to CCR4 more strongly than TARC (J. Biol. Chem., 273, 1764 (1998)).
It has been shown that CCR4 is expressed on CD4+Th cells which produce cytokine and/or chemokine (J. Biol. Chem., 272, 15036 (1997)), and it has been reported that CCR4 is expressed selectively on Th2 cells among CD4+Th cells (J. Exp. Med. 187, 129 (1998), J. Immunol., 161, 5111 (1998)). In addition, CCR4+ cells have been found in effector/memory T cells (CD4+/CD45RO+), and when CCR4+ cells were stimulated, IL-4 and IL-5 were produced but IFN-γ was not produced (Int. Immunol., 11, 81 (1999)). Also, it has been reported that CCR4+ cells belong to a CLA (cutaneous lymphocyte antigen)-positive and α4β8 integrin-negative group among memory T cells, and CCR4 is expressed on memory T cells related not to gut immunity but to systemic immunity of the skin and the like (Nature, 400, 776 (1999)) These results strongly suggest that when inflammation is induced, the memory T cells are activated to express CCR4 and ate migrated into the inflammatory site by MDC and TARC of ligands of CCR4, and accelerate activation of other inflammatory cells.
It has been recently found that CCR4 is also expressed in natural killer cells (Journal of Immunology, 164, 4048-4054 (200), Blood, 97, 367-375 (2001)) and platelets (Thrombosis Research, 101, 279-289 (2001), Blood, 96, 4046-4054 (2000), Blood, 97, 937-945 (2001)) in human.
It is known that an antagonist of TARC or MDC as a ligand of CCR4, namely a CCR4 antagonist, inhibits platelet aggregation (WO 99/15666). It is known that such an agent modulating the function of CCR4 also affects on platelet functions.
Expression of CCR4 is found in platelets. For example, Adrian et al. (Blood, 97, 937-945 (2001)) and Abi-Younes et al. (Thrombosis Research, 101, 279-289 (2001), WO 00/42074, WO 00/41724) have detected expression of CCR4 in human platelets using anti-CCR4 antibodies. An antibody having reactivity with CCR4 but not capable of binding to human platelets has not been known to date.
Also, it is known that when an autoantibody capable of binding to platelets is produced, autoimrnune thrombopenia is induced (Blood, 70, 428-431 (1987), Transfusion Science, 19, 245-251 (1998)). Agents having influences on thrombocytopenia and platelet functions are not generally desirable as medicaments because they often cause severe side effects such as bleeding and thrombus formation. Particularly, since antibodies have long blood half-life, an antibody which affects on platelet functions is difficult to be developed as a medicament. For example, development of anti-CD40 ligand antibodies which had been developed as agents for treating autoirnrnune diseases has been suspended because of the generation of side effects which are considered to be due to recognition of antigen expressed on activated platelets (Nature Medicine, 6, 114 (2000), BioCentury, A8 of 18 (2002.06.20)).
It is known that proteins are subjected to various modification reactions after translation. One of the modification reactions known is a sulfated reaction of tyrosine residues. It has been reported that many proteins are sulfated at tyrosine residues (Chemistry and Biology, 7, R57-R61 (2000)). The tyrosine residue which is sulfated has characteristic that many acidic amino acid residues are present in its vicinity, and a protein having a possibility of being sulfated and its region have been suggested (Cell, 96, 667-676 (1999)). Regarding CCR4, 4 tyrosine residues are present close to the N-terminal, but there are no reports showing that the tyrosine residues are sulfated.
As the current method for treating Th2-rediated imnune diseases, the followings have been developed: (1) antagonists for cytokines and chemokines such as a humanized anti-IL-5 antibody (SB-240563: Smith Kline Beecharm, Sch-55700 (CDP-835): Shehling Plough/Celitech), a humanized IL-4 antibody (U.S. Pat. No. 5,914,110), a soluble chemokine receptor (J. Immunol., 160, 624 (1998)), etc.; (2) cytokine/chemokine production inhibitors such as an IL-5 production inhibitor (Japanese Published Unexamined Patent Application No. 53355/96), a retinoid antagonist (WO 99/24024), splatast tosilate (IPD-1151 T, manufactured by Taiho Pharmaceutical), etc.; (3) agents acting on eosinophil, mast cell and the like as final inflammatory cells, such as a humanized IL-5 receptor antibody (WO 97/10354), a CC chemokine receptor 3 (CCR3) antagonist (Japanese Published Unexamined Patent Application No. 147872/99), etc.; (4) inflammatory molecule inhibitors such as a humanized anti-IgE antibody (Am. J. Respir. Crit. Care Med., 157, 1429 (1998)), etc.; and the like. But they inhibit only a part of the elaborate network among cytokine, chemokine and inflammatory cells. Th2-mediated immune diseases should not be cured by these agents Anti-CD4 antibodies have an activity to control T cells, and have effects on steroid-dependent severe asthma. However, since the CD4 molecule is broadly expressed in various immune cells, they lack in specificity and have a drawback of accompanying strong immunosuppressive effect (Int. Arch. Aller. Immunol., 118, 133 (1999)).
Thus, in order to inhibit all of them, it is required to control specifically upstream of the problematic allergic reaction, namely Th2 cells.
The currently used common method for treating patients of severe Th2-mediated immune diseases is steroid administration, but side effects by steroids cannot be avoided. Also, there are drawbacks that the conditions of each patient return to the former state when the steroid administration is suspended, and that drug resistance is acquired when the steroid is administered for a long time.
To date, no human CDR-grafted antibody and the antibody fragment thereof which can detect CCR4-expressing cells and also has cytotoxicity against CCR4-expressing cells has been established. In addition, no therapeutic agent which can inhibit production of Th2 cytokine has been known so far.
Although it has been reported that CCR4 is also expressed on the cancer cells of leukemia patients (Blood, 96, 685 (2000)), no therapeutic agent which depletes leukemia cells has been known.
It is known in general that when an antibody derived from a non-human animal, e.g., a mouse antibody, is administered to human, it is recognized as an foreign substance and induces a human antibody against the mouse antibody (human anti-mouse antibody: hereinafter referred to as “HAMA”) in the human body It is known that the HAMA reacts with the administered mouse antibody to cause side effects (J. Clin. Oncol., 2, 881 (1984), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80, 932 (1988), Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)), to accelerate disappearance of the administered mouse antibody from the body (J. Nucl Med., 26, 1011 (1985), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80, 937 (1988)), and to reduce therapeutic effects of the mouse antibody (J. Immunol., 135, 1530 (1985), Cancer Res., 46, 6489 (1986)).
In order to solve these problems, attempts have been made to convert an antibody derived from a non-human animal into a human CDR-grafted antibody using genetic engineering technique.
The human CDR-grafted antibody is an antibody in which the amino acid sequence of CDR in the variable region (hereinafter referred to as “V region”) derived from a non-human animal antibody is grafted into an appropriate position of a human antibody (Nature, 321, 522 (1986)). In comparison with antibodies derived from non-human animals such as mouse antibodies and the like, these human CDR-grafted antibodies have various advantages for clinical applications to human. For example, it has been reported that its immmunogenecity was reduced and its blood half-life became long compared with a mouse antibody using a monkey (Cancer Res., 56, 1118 (1996), Immunol., 85, 668 (1995)). Thus, it is expected that the human CDR-grafted antibodies have less side effects in human and their therapeutic effects continue for a longer time than antibodies derived from non-human animals.
Furthermore, since the human CDR-grafted antibody is prepared by using genetic engineering technique, molecules in various forms can be prepared For example, when γl subclass is used as a heavy chain (hereinafter referred to as “H chain”) constant region (hereinafter referred to as “C region”) (H chain C region is referred to as “CH”) of a human antibody, a humanized antibody having a high effector function such as antibody-dependent cell-mediated cytotoxic (hereinafter referred to as “ADCC”) activity can be prepared (Cancer Res., 56, 1118 (1996)) and a prolonged blood half-life compared with a mouse antibody is expected (Immunol., 85, 668 (1995)). Also, in treatment particularly for reducing the number of CCR4-expressing cells, higher cytotoxic activities such as complement-dependent cytotoxic activity (hereinafter referred to as “CDC activity”) and ADCC activity via the Fc region (the region in and after the hinge region of an antibody heavy chain) of an antibody are important for the therapeutic effects. Therefore, these results clearly show that human CDR-grafted antibodies are preferred to antibodies derived from non-human animals such as mouse antibodies.
Furthermore, according to the recent advances in protein engineering and genetic engineering, antibody fragments having a smaller molecular weight such as Fab, Fab′, F(ab′)2, a single chain antibody (hereinafter referred to as “scFv”) (Science, 242, 423 (1988)), a dimerized V region fragment (hereinafter referred to as “Diabody”) (Nature Biotechnol., 15, 629 (1997)), a disulfide stabilized V region fragment (hereinafter referred to as “dsFv”) (Molecular Immunol, 32, 249 (1995)), a peptide containing CDR (J. Biol Chem., 271, 2966 (1996)) and the like, can be prepared as human CDR-grafted antibodies. The antibody fragments are excellent in transitional activity into target tissues compared to complete antibody molecules (Cancer Res., 52, 3402 (1992)).
It is considered that these fragments derived from human CDR-grafted antibodies and antibody fragments thereof are more desirable than those derived from antibodies derived from non-human animals such as mouse antibodies, when used in clinical applications to human.
As described above, diagnostic and therapeutic effects can be expected from human CDR-grafted antibodies and antibody fragments thereof when used alone, but attempts have been made to further improve the effects by using other molecules in combination. For example, cytokine can be used as one of such molecules. Cytokine is a general term for various soluble factors which control intercellular mutual functions in immune reactions. CDC activity and ADCC activity, for example, are known as the cytotoxic activities of antibodies, and ADCC activity is controlled by effector cells having Fc receptors on the cell surface such as monocytes, macrophages, NK cells and the like (J. Immunol., 138, 1992 (1987)). Since various cytokines activate these effector cells, they can be administered in combination with an antibody in order to improve ADCC activity of the antibody and the like.